Passive Swelling Research Articles

Role of Side-Chain Free Volume on the Electrochemical Behavior of Poly(propylenedioxythiophenes).

Mixed ionic/electronic conducting polymers are versatile systems for, e.g., energy storage, heat management (exploiting electrochromism), and biosensing, all of which require electrochemical doping, i.e., the electrochemical oxidation or reduction of their macromolecular backbones. Electrochemical doping is achieved via electro-injection of charges (i.e., electronic carriers), stabilized via migration of counterions from a supporting electrolyte. Since the choice of the polymer side-chain functionalization influences electrolyte and/or ion sorption and desorption, it in turn affects redox properties, and, thus, electrochemically induced mixed conduction. However, our understanding of how side-chain versus backbone design can increase ion flow while retaining high electronic transport remains limited. Hence, heuristic design approaches have typically been followed. Herein, we consider the redox and swelling behavior of three poly(propylenedioxythiophene) derivatives, P(ProDOT)s, substituted with different side-chain motifs, and demonstrate that passive swelling is controlled by the surface polarity of P(ProDOT) films. In contrast, active swelling under operando conditions (i.e., under an applied bias) is dictated by the local side-chain free volume on the length scale of a monomer unit. Such insights deliver important design criteria toward durable soft electrochemical systems for diverse energy and biosensing platforms and new understanding into electrochemical conditioning ("break-in") in many conducting polymers.

Mapping the nanoscale elastic property modulations of polypyrrole thin films in liquid electrolyte with EC-AFM.

Linking structure to mechanical and elastic properties is a major concern for the development of novel electroactive materials. This work reports on the potential-induced changes in thickness and Young modulus of a substrate supported, perchlorate doped polypyrrole thin film (<100 nm) investigated with electrochemical atomic force microscopy (AFM) under in situ conditions. This was accomplished by nanomechanical mapping of potentiodynamically electropolymerized polypyrrole film in electrolyte solution with AFM during redox cycling. The polypyrrole film thickness and Young modulus follow the electrical potential nearly linearly, increasing due to solvent and ion influx as the film is oxidized, and decreasing during reduction. Our measurements also confirm the presence of a potential-independent, passive swelling which is accompanied by softening of the film, likely caused by osmotic effects. Additionally, the heterogeneous distribution of the Young modulus can be directly traced to the typical nodular surface topography of polypyrrole, with the top of the nodular area possessing lower modulus, thus highlighting the complex relationship between topography and elastic properties.

Toward Measuring the Mechanical Stresses Exerted by Branching Embryonic Airway Epithelial Explants in 3D Matrices of Matrigel.

Numerous organs in the bodies of animals, including the lung, kidney, and mammary gland, contain ramified networks of epithelial tubes. These structures arise during development via a process known as branching morphogenesis. Previous studies have shown that mechanical forces directly impact this process, but the patterns of mechanical stress exerted by branching embryonic epithelia are not well understood. This is, in part, owing to a lack of experimental tools. Traditional traction force microscopy assays rely on the use of compliant hydrogels with well-defined mechanical properties. Isolated embryonic epithelial explants, however, have only been shown to branch in three-dimensional matrices of reconstituted basement membrane protein, or Matrigel, a biomaterial with poorly characterized mechanical behavior, especially in the regime of large deformations. Here, to compute the traction stresses generated by branching epithelial explants, we quantified the finite-deformation constitutive behavior of gels of reconstituted basement membrane protein subjected to multi-axial mechanical loads. We then modified the mesenchyme-free assay for the ex vivo culture of isolated embryonic airway epithelial explants by suspending fluorescent microspheres within the surrounding gel and tracking their motion during culture. Surprisingly, the tracked bead motion was non-zero in regions of the gel far away from the explants, suggestive of passive swelling deformations within the matrix. To compute accurate traction stresses, these swelling deformations must be decomposed from those generated by the branching explants. We thus tracked the motion of beads suspended within cell-free matrices and quantified spatiotemporal patterns of gel swelling. Taken together, these passive swelling data can be combined with the measured mechanical properties of the gel to compute the traction forces exerted by intact embryonic epithelial explants.

In Situ Studies of the Swelling by an Electrolyte in Electrochemical Doping of Ethylene Glycol-Substituted Polythiophene.

Organic mixed ionic electronic conductors (OMIECs) have the potential to enable diverse new technologies, ranging from biosensors to flexible energy storage devices and neuromorphic computing platforms. However, a study of these materials in their operating state, which convolves both passive and potential-driven solvent, cation, and anion ingress, is extremely difficult, inhibiting rational material design. In this report, we present a novel approach to the in situ studies of the electrochemical switching of a prototypical OMIEC based on oligoethylene glycol (oEG) substitution of semicrystalline regioregular polythiophene via grazing-incidence X-ray scattering. By studying the crystal lattice both dry and in contact with the electrolyte while maintaining potential control, we can directly observe the evolution of the crystalline domains and their relationship to film performance in an electrochemically gated transistor. Despite the oEG side-chain enabling bulk electrolyte uptake, we find that the crystalline regions are relatively hydrophobic, exhibiting little (less than one water per thiophene) swelling of the undoped polymer, suggesting that the amorphous regions dominate the reported passive swelling behavior. With applied potential, we observe that the π-π separation in the crystals contracts while the lamella spacing increases in a balanced fashion, resulting in a negligible change in the crystal volume. The potential-induced changes in the crystal structure do not clearly correlate to the electrical performance of the film as an organic electrochemical transistor, suggesting that the transistor performance is strongly influenced by the amorphous regions of the film.

The modes and competing rates of cartilage fluid loss and recovery

Cartilage loses, recovers, and maintains its thickness, hydration, and biomechanical functions based on competing rates of fluid loss and recovery under varying joint-use conditions. While the mechanics and implications of load-induced fluid loss have been studied extensively, those of fluid recovery have not. This study isolates, quantifies, and compares rates of cartilage recovery from three known modes: (1) passive swelling – fluid recovery within a static unloaded contact area; (2) free swelling – unrestricted fluid recovery by an exposed surface; (3) tribological rehydration – fluid recovery within a loaded contact area during sliding. Following static loading of adult bovine articular cartilage to between 100 and 500 μm of compression, passive swelling, free swelling, and tribological rehydration exhibited average rates of 0.11 ± 0.04, 0.71 ± 0.15, and 0.63 ± 0.22 μm/s, respectively, over the first 100 s of recovery; for comparison, the mean exudation rate just prior to sliding was 0.06 ± 0.04 μm/s. For this range of compressions, we detected no significant difference between free swelling and tribological rehydration rates. However, free swelling and tribological rehydration rates, those associated with joint articulation, were ∼7-fold faster than passive swelling rates. While previous studies show how joint articulation prevents fluid loss indefinitely, this study shows that joint articulation reverses fluid loss following static loading at >10-fold the preceding exudation rate. These competitive recovery rates suggest that joint space and function may be best maintained throughout an otherwise sedentary day using brief but regular physical activity. Statement of SignificanceCartilage loses, recovers, and maintains its thickness, hydration, and biomechanical functions based on competing rates of fluid loss and recovery under varying joint-use conditions. While load-induced fluid loss is extremely well studied, this is the first to define the competing modes of fluid recovery and to quantify their rates. The results show that the fluid recovery modes associated with joint articulation are 10-fold faster than exudation during static loading and passive swelling during static unloading. The results suggest that joint space and function are best maintained throughout an otherwise sedentary day using brief but regular physical activities.

The Modes and Competing Rates of Cartilage Fluid Loss and Recovery

Cartilage loses, recovers, and maintains its thickness, hydration, and biomechanical functions by adjusting relative rates of load-induced fluid loss and competitive fluid recovery under varying joint-use conditions. While the mechanics and implications of fluid loss have been studied extensively, those of fluid recovery have not. This study isolates, quantifies, and compares rates of cartilage recovery from three known modes: (1) passive swelling – fluid recovery within a nominally unloaded contact; (2) free swelling – fluid recovery at an exposed surface; (3) tribological rehydration – fluid recovery within a loaded contact area during sliding. Following static loading of adult bovine articular cartilage to between 100 and 250 μm of compression, passive swelling, free swelling, and tribological rehydration rates were 0.18 ± 0.08 μm/s, 1.7 ± 0.3, and 1.8 ± 0.6 μm/s, respectively; the corresponding exudation rate was 0.06 ± 0.04 μm/s. For this range of compressions, we detected no significant difference between passive swelling and exudation rates. However, free swelling and tribological rehydration rates, those associated with joint articulation, were 10-fold faster than passive swelling and exudation rates. While previous studies show how joint articulation prevents fluid loss indefinitely, this study shows how quickly joint articulation reverses fluid loss following static loading (at 30:1 the exudation rate). These competitive recovery rates suggest that joint space and function may be best maintained throughout an otherwise sedentary day using brief but regular physical activities (e.g. a 5-minute walk).

A Low-Swelling Polymeric Mixed Conductor Operating in Aqueous Electrolytes.

Organic mixed conductors find use in batteries, bioelectronics technologies, neuromorphic computing, and sensing. While great progress has been achieved, polymer-based mixed conductors frequently experience significant volumetric changes during ion uptake/rejection, i.e., during doping/de-doping and charging/discharging. Although ion dynamics may be enhanced in expanded networks, these volumetric changes can have undesirable consequences, e.g., negatively affecting hole/electron conduction and severely shortening device lifetime. Here, the authors present a new material poly[3-(6-hydroxy)hexylthiophene] (P3HHT) that is able to transport ions and electrons/holes, as tested in electrochemical absorption spectroscopy and organic electrochemical transistors, and that exhibits low swelling, attributed to the hydroxylated alkyl side-chain functionalization. P3HHT displays a thickness change upon passive swelling of only +2.5%, compared to +90% observed for the ubiquitous poly(3,4-ethylenedioxythiophene):polystyrene sulfonate, and +10 to +15% for polymers such as poly(2-(3,3'-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-[2,2'-bithiophen]-5-yl)thieno[3,2-b]thiophene) (p[g2T-TT]). Applying a bias pulse during swelling, this discrepancy becomes even more pronounced, with the thickness of P3HHT films changing by <10%while that of p(g2T-TT) structures increases by +75 to +80%. Importantly, the initial P3HHT film thickness is essentially restored after de-doping while p(g2T-TT) remains substantially swollen. The authors, thus, expand the materials-design toolbox for the creation of low-swelling soft mixed conductors with tailored properties and applications in bioelectronics and beyond.

Effects of Gd3+ and Ca2+ on Frog Heart Muscle Contractility and Respiration, Swelling and Inner Membrane Potential of Rat Heart Mitochondria

We studied the inotropic and chronotropic effects of gadolinium ions (Gd3+) on contractility of frog Rana ridibunda heart muscle preparations. In addition, the influence of Gd3+ on oxygen uptake rates, swelling and the mitochondrial inner membrane potential (ΔΨmito) were studied in glutamate- and malate-energized rat heart mitochondria (RHM). It was found that Gd3+ decreases the amplitude and frequency of spontaneous heart contractions. At the same time, Gd3+ prevents Ca2+-induced swelling of these organelles in saline media and falling of ΔΨmito. In this case, regardless of the presence of Ca2+ in the medium, Gd3+ has a weak effect on mitochondrial state 3 or 3UDNP respiration (in the presence of 2,4-dinitrophenol). In calcium free experiments, Gd3+ stimulates passive swelling of RHM. These effects of Gd3+ may indicate that Gd3+ has no toxic effect on RHM but, on the other hand, inhibits opening of the mitochondrial permeability transition pore (MPTP) in the inner membrane. Our data provide better insight into the mechanisms of action of rare earth elements on Ca2+-dependent processes in the myocardium of vertebrates.

ВЛИЯНИЕ Gd 3+ И Ca 2+ НА СОКРАТИМОСТЬ СЕРДЕЧНОЙ МЫШЦЫ ЛЯГУШКИ И ДЫХАНИЕ, НАБУХАНИЕ И ПОТЕНЦИАЛ ВНУТРЕННЕЙ МЕМБРАНЫ МИТОХОНДРИЙ СЕРДЦА КРЫСЫ

The inotropic and chronotropic effects of gadolinium ions (Gd3+) were studied in heart muscle preparations of the frog Rana ridibunda. In addition, the influence of Gd3+ on oxygen consumption rates, swelling and the inner membrane potential (∆Ψmito) were studied in glutamate and malate energized rat heart mitochondria (RHM). It was found that Gd3+ decreases the amplitude and frequency of spontaneous heart contractions. At the same time, Gd3+ prevents Ca2+ -induced swelling of these organelles in salt media and falling ∆Ψmito. In this case, regardless of the presence of Ca2+ in the medium, Gd3+ has a weak effect on mitochondrial state 3 or 3UDNP respiration (in the presence of 2,4-dinitrophenol). In calcium free experiments, Gd3+ stimulates passive swelling of RHM. These effects of Gd3+ may indicate that Gd3+ does not exert a toxic effect on RHM but, on the other hand, inhibits opening of the mitochondrial permeability transition pore (MPTP) in the inner membrane. Our data provide better insight into the mechanisms of action of rare earth elements on Ca2+ -dependent processes in the myocardium of vertebrates.

Kinematical, Structural and Mechanical Adaptations to Desiccation in Poikilohydric Ramonda myconi (Gesneriaceae).

Resurrection plants have fascinated scientists since centuries as they can fully recover from cellular water contents below 10%, concomitantly showing remarkable leaf folding motions. While physiological adaptations have been meticulously investigated, the understanding of structural and mechanical adaptations of this phenomenon is scarce. Using imaging and bending techniques during dehydration-rehydration experiments, morphological, anatomical, and biomechanical properties of desiccation-tolerant Ramonda myconi are examined, and selected structural adaptations are compared to those of homoiohydrous Monophyllaea horsfieldii (both Gesneriaceae). At low water availability, intact and cut-off R. myconi leaves undergo considerable morphological alterations, which are fully and repeatedly reversible upon rehydration. Furthermore, their petioles show a triphasic mechanical behavior having a turgor-based structural stability at high (Phase 1), a flexible mechanically state at intermediate (Phase 2) and a material-based stability at low water contents (Phase 3). Lastly, manipulation experiments with cut-off plant parts revealed that both the shape alterations of individual structures, as well as, the general leaf kinematics largely rely on passive swelling and shrinking processes. Taken together, R. myconi possesses structural and mechanical adaptations to desiccation (in addition to physiological adaptations), which may mainly be passively driven by its water status influenced by the water fluctuations in its surroundings.

Uncoupling proteins UCP2 and UCP3 from mitochondria of liver and skeletal muscle of ground squirrel Spermophilus undulatus do not transport pyruvate in contrast to UCP1 from brown adipose tissue

Physiological role of mitochondrial uncoupling proteins UCP2 and UCP3, homologous to UCP1 from brown adipose tissue, is unclear. It was proposed recently that UCP2 and UCP3 are metabolic triggers that switch oxidation of glucose to oxidation of fatty acids, exporting pyruvate from mitochondria. In the present study we tried to verify this hypothesis using ground squirrels (Spermophilus undulatus), since expression of all UCPs in different tissues increases during winter season, and UCP1 is abundant in brown fat. We confirmed the possibility of nonspecific transport of pyruvate through UCP1 in brown fat mitochondria and tried to identify similar transport in liver and skeletal muscle mitochondria where UCP2 and UCP3 are expressed. Transport of pyruvate mediated by UCP1 in mitochondria of brown fat was observed using valinomycin-induced swelling of non-respiring mitochondria in 55 mM potassium pyruvate and was inhibited by GDP. In contrast, mitochondria of liver and skeletal muscles in similar conditions did not exhibit electrogenic transport of pyruvate anions that could be related to functioning of UCP2 and UCP3. At the same time, functioning of pyruvate carrier was detected in these mitochondria by nigericin-induced passive swelling or valinomycin-induced active swelling in potassium pyruvate that was inhibited by α-CHC, a specific inhibitor of the pyruvate carrier. Thus, our results suggest that in contrast to UCP1 of brown fat, UCP2 and UCP3 from intact liver and skeletal muscle mitochondria of winter active ground squirrels are unable to carry out pyruvate transport.

Elasticity of the Porcine Lens Capsule as Measured by Osmotic Swelling

As an alternative to purely mechanical methods, optical tracking of passive osmotic swelling was used to assess mechanical properties of the porcine lens capsule. A simple model was developed accounting for the permeability of the lens fiber cells and capsule to water, the concentration of fixed charges in the fiber cells, and the capsule's resistance to the swelling of fiber cells. Fitting the model solution to experimental data provided an estimate of the elastic modulus of the lens capsule under the assumption of linear isotropic elasticity. The calculated elastic modulus at a fixed charge density of 20 mol m(-3) was 2.0+/-0.5 MPa (mean+/-95% confidence interval; n=15) for 0.1% saline solution, 0.64+/-0.3 MPa (n=10) for 0.2% saline solution, and 0.28+/-0.5 MPa (n=6) for 0.5% saline solution. These values are comparable to previously reported moduli of elasticity for the porcine lens capsule at small strains (<10%), and the slight increase with hypotonicity is consistent with the nonlinear mechanical behavior of the lens capsule. Although limited by being a single measurement on a heterogeneous tissue, osmotic swelling provides a quantitative assessment of the stiffness of the lens capsule without requiring dissection or manipulation of the lens. Thus, the new method could be useful for small animal models.

NHA-oc/NHA2: A mitochondrial cation–proton antiporter selectively expressed in osteoclasts

Bone resorption is regulated by a complex system of hormones and cytokines that cause osteoblasts/stromal cells and lymphocytes to produce factors including RANKL, that ultimately result in the differentiation and activation of osteoclasts, the bone resorbing cells. We used a microarray approach to identify genes upregulated in RANKL-stimulated osteoclast precursor cells. Osteoclast expression was confirmed by multiple tissue Northern and in situ hybridization analysis. Gene function studies were carried out by siRNA analysis. We identified a novel gene, which we termed nha-oc/NHA2, which is strongly upregulated during RANKL-induced osteoclast differentiation in vitro and in vivo. nha-oc/NHA2 encodes a novel cation–proton antiporter (CPA) and is the mouse orthologue of a human gene identified in a database search: HsNHA2. nha-oc/NHA2 is selectively expressed in osteoclasts. NHA-oc/NHA2 protein localizes to the mitochondria, where it mediates Na +-dependent changes in mitochondrial pH and Na + acetate induced mitochondrial passive swelling. RNA silencing of nha-oc/nha2 reduces osteoclast differentiation and resorption, suggesting a role for NHA-oc/NHA2 in these processes. nha-oc/NHA2 therefore is a novel member of the CPA family and is the first mitochondrial NHA characterized to date. nha-oc/NHA2 is also unique in that it is the first eukaryotic and tissue-specific CPA2 characterized to date. NHA-oc/NHA2 displays the expected activities of a bona fide CPA and plays a key role(s) in normal osteoclast differentiation and function.

A Mechano-chemical Model for the Passive Swelling Response of an Isolated Chondron under Osmotic Loading

The chondron is a distinct structure in articular cartilage that consists of the chondrocyte and its pericellular matrix (PCM), a narrow tissue region surrounding the cell that is distinguished by type VI collagen and a high glycosaminoglycan concentration relative to the extracellular matrix. We present a theoretical mechano-chemical model for the passive volumetric response of an isolated chondron under osmotic loading in a simple salt solution at equilibrium. The chondrocyte is modeled as an ideal osmometer and the PCM model is formulated using triphasic mixture theory. A mechano-chemical chondron model is obtained assuming that the chondron boundary is permeable to both water and ions, while the chondrocyte membrane is selectively permeable to only water. For the case of a neo-Hookean PCM constitutive law, the model is used to conduct a parametric analysis of cell and chondron deformation under hyper- and hypo-osmotic loading. In combination with osmotic loading experiments on isolated chondrons, model predictions will aid in determination of pericellular fixed charge density and its relative contribution to PCM mechanical properties.

Mitochondrial transport in proline catabolism in plants: the existence of two separate translocators in mitochondria isolated from durum wheat seedlings

Abiotic stresses, such as high salinity or drought, can cause proline accumulation in plants. Such an accumulation involves proline transport into mitochondria where proline catabolism occurs. By using durum wheat seedlings as a plant model system, we investigated how proline enters isolated coupled mitochondria. The occurrence of two separate translocators for proline, namely a carrier solely for proline and a proline/glutamate antiporter, is shown in a functional study in which we found the following: (1) Mitochondria undergo passive swelling in isotonic proline solutions in a stereospecific manner. (2) Externally added L: -proline (Pro) generates a mitochondrial membrane potential (Delta Psi) with a rate depending on the transport of Pro across the mitochondrial inner membrane. (3) The dependence of the rate of generation of Delta Psi on increasing Pro concentrations exhibits hyperbolic kinetics. Proline transport is inhibited in a competitive manner by the non-penetrant thiol reagent mersalyl, but it is insensitive to the penetrant thiol reagent N-ethylmaleimide (NEM). (4) No accumulation of proline occurs inside the mitochondria as a result of the addition of proline externally, whereas the content of glutamate increases both in mitochondria and in the extramitochondrial phase. (5) Glutamate efflux from mitochondria occurs at a rate which depends on the mitochondrial transport, and it is inhibited in a non-competitive manner by NEM. The dependence of the rate of glutamate efflux on increasing proline concentration shows saturation kinetics. The physiological role of carrier-mediated transport in the regulation of proline catabolism, as well as the possible occurrence of a proline/glutamate shuttle in durum wheat seedlings mitochondria, are discussed.

Properties of the Permeability Transition Pore in Mitochondria Devoid of Cyclophilin D

We have studied the properties of the permeability transition pore (PTP) in mitochondria from the liver of mice where the Ppif gene encoding for mitochondrial Cyclophilin D (CyP-D) had been inactivated. Mitochondria from Ppif-/- mice had no CyP-D and displayed a striking desensitization of the PTP to Ca2+, in that pore opening required about twice the Ca2+ load necessary to open the pore in strain-matched, wild-type mitochondria. Mitochondria lacking CyP-D were insensitive to Cyclosporin A (CsA), which increased the Ca2+ retention capacity only in mitochondria from wild-type mice. The PTP response to ubiquinone 0, depolarization, pH, adenine nucleotides, and thiol oxidants was similar in mitochondria from wild-type and Ppif-/- mice. These experiments demonstrate that (i) the PTP can form and open in the absence of CyP-D, (ii) that CyP-D represents the target for PTP inhibition by CsA, and (iii) that CyP-D modulates the sensitivity of the PTP to Ca2+ but not its regulation by the proton electrochemical gradient, adenine nucleotides, and oxidative stress. These results have major implications for our current understanding of the PTP and its modulation in vitro and in vivo.

Passive penetration of nitrate through the plasma membrane of Paracoccus denitrificans and its potentiation by the lipophilic tetraphenylphosphonium cation

Previously, it has been shown that treatment of Paracoccus denitrificans cells with phenylglyoxal inhibits the methyl-viologen-linked nitrate reductase activity by blocking the nitrate transporter. This inhibition disappears if tetraphenylphosphonium cation (TPP +) is added to the assay medium. In the present paper, the following evidence suggests that the effect of TPP + results from an increased transmembrane anion permeability and not from transporter reactivation or cell lysis. (1) Beside nitrate, TPP + also mediated the utilisation of chlorate, which normally lacks access to the cytoplasm. (2) The TPP + pathway had about hundred-times higher K m values for nitrate and chlorate than nitrate reductase in Triton X-100 permeabilised cells. (3) Although the uncoupler CCCP alone failed to overcome the PG block, it stimulated the operation of the TPP + pathway. (4) The method of continuous variations allowed the transport stoichiometry TPP +/NO 3 − to be determined as 3, indicating charge compensation for nitrate movement and the subsequent transmembrane two-electron redox reaction. Anion uptake was also measured independently from passive swelling of uncoupled spheroplasts in iso-osmotic solutions of ammonium salts. The permeability to nitrate lay in the permeability sequence Cl −<NO 3 −<ClO 4 −<SCN − and was further enhanced by TPP +.

The uncoupling effect of the nonsteroidal anti-inflammatory drug nimesulide in liver mitochondria from adjuvant-induced arthritic rats.

The aim of the present study was to evaluate the changes caused by adjuvant-induced arthritis in liver mitochondria and to investigate the effects of the nonsteroidal anti-inflammatory drug nimesulide. The main alterations observed in liver mitochondria from arthritic rats were: higher rates of state IV and state III respiration with beta-hydroxybutyrate as substrate; reduced respiratory control ratio and impaired capacity for swelling dependent on beta-hydroxybutyrate oxidation. No alterations were found in the activities of NADH oxidase and ATPase. Nimesulide produced: (1) stimulation of state IV respiration; (2) decrease in the ADP/O ratio and in the respiratory control ratio; (3) stimulation of ATPase activity of intact mitochondria; (4) inhibition of swelling driven by the oxidation of beta-hydroxybutyrate; (5) induction of passive swelling due to NH(3)/NH(4)+ redistribution. The activity of NADH oxidase was insensitive to nimesulide. Mitochondria from arthritic rats showed higher sensitivity to nimesulide regarding respiratory activity. The results of this work allow us to conclude that adjuvant-induced arthritis leads to quantitative changes in some mitochondrial functions and in the sensitivity to nimesulide. Direct evidence that nimesulide acts as an uncoupler was also presented. Since nimesulide was active in liver mitochondria at therapeutic levels, the impairment of energy metabolism could lead to disturbances in the liver responses to inflammation, a fact that should be considered in therapeutic intervention.

Activation of ion-conducting pathways in the inner mitochondrial membrane – an unrecognized activity of fatty acid?

The effect of non-esterified myristate (C14:0) or dodecyl sulfate was studied on passive swelling of rat liver mitochondria suspended in hypotonic alkaline KCl medium in the absence of the potassium ionophore valinomycin. Both compounds rapidly initiated large-amplitude swelling. However, they failed to initiate swelling when the mitochondria were suspended in hypotonic alkaline sucrose medium. In contrast to myristate or dodecyl sulfate, the non-ionic detergent Triton X-100 initiated swelling of mitochondria in both of the media. The following findings indicate that the inner mitochondrial membrane (IMM) is permeabilized by myristate to K + and Cl − in a specific manner. (i) Swelling initiated by myristate did not respond to cyclosporin A, (ii) the protonophoric uncoupler FCCP was unable to mimic the myristate effect on swelling, and (iii) myristate-induced Cl −-permeation (measured with KCl medium plus valinomycin) was inhibited by N, N′-dicyclohexylcarbodiimide, quinine or ATP. Myristate- or dodecyl sulfate-initiated swelling was paralleled by the lowering of endogenous Mg 2+ content. Both effects, stimulation of swelling and depletion of endogenous Mg 2+ are correlated with each other. Similar effects have been reported previously for the carboxylic divalent cation ionophore calcimycin (A23187). The A23187-induced swelling has identical inhibiting characteristics on Cl −-permeation with respect to N, N′-dicyclohexylcarbodiimide, quinine and ATP as the myristate-stimulated swelling. Therefore, we conclude that non-esterified fatty acids increase the permeability of mitochondria to K + and Cl − at alkaline pH by activating Mg 2+-dependent ion-conducting pathways in IMM.

Matrix free Mg(2+) and the regulation of mitochondrial volume.

Mitochondria must maintain volume homeostasis in order to carry out oxidative phosphorylation. It has been postulated that the concentration of free Mg(2+) ([Mg(2+)]) serves as the sensor of matrix volume and regulates a K(+)-extruding K(+)/H(+) antiport (K. D. Garlid. J. Biol. Chem. 255: 11273-11279, 1980). To test this hypothesis, the fluorescent probe furaptra was used to monitor [Mg(2+)] and free Ca(2+) concentration ([Ca(2+)]) in the matrix of isolated beef heart mitochondria, and K(+)/H(+) antiport activity was measured by passive swelling in potassium acetate. Concentrations that result in 50% inhibition of maximum activity of 92 microM matrix [Mg(2+)] and 2.2 microM [Ca(2+)] were determined for the K(+)/H(+) antiport. Untreated mitochondria average 670 microM matrix [Mg(2+)], a value that would permit <1% of maximum K(+)/H(+) antiport activity. Hypotonic swelling results in large decreases in matrix [Mg(2+)], but swelling due to accumulation of acetate salts does not alter [Mg(2+)]. Swelling in phosphate salts decreases matrix [Mg(2+)], but not to levels that permit appreciable antiport activity. We conclude that 1) it is unlikely that matrix [Mg(2+)] serves as the mitochondrial volume sensor, 2) if K(+)/H(+) antiport functions as a volume control transporter, it is probably regulated by factors other than [Mg(2+)], and 3) alternative mechanisms for mitochondrial volume control should be considered.